Developing cancers are actively guided by their microenvironments, and at the same time they influence those surroundings. During metastatic invasion, the stromal cells that surround the cancer cells become a local "invasion field," whose micro-vascularization and extracellular matrices differ from normal. Because changes in microenvironment occur relatively rapidly, they have most often been attributed to secreted (indirect) factors exchanged between the two cell populations. Direct cell-cell communication, however, including cytoplasmic channels known as gap junctions, must not be overlooked as a regulatory mechanism during cancer progression. Recent studies of prostate cancer in our laboratory, together with others' work on several carcinomas, reveal increases in the direct gap junctional connection between cancer and stromal cells during cancer progression, as well as increases in the expression levels of specific connexin protein isoforms (out of which the gap junctions are constructed). Evidence is now accumulating that profiles of connexin proteins are critical for invasive behavior. How connexins are regulated during aging, cancer progression and invasion, and how connexins themselves regulate cell behaviors are the focuses of this proposal. We have found that androgen levels (which change as men age) affect connexin profiles, that these profiles affect cell invasion into stromal cell organoids, and that connexins are able to complex with Focal Adhesion Kinase (FAK), a molecule long-known to regulate cell motility via integrin cell adhesion. We aim: 1) to further characterize regulatory effects of cell co-culture, conditioned media, and androgens on connexin expression profiles in cell lines representing different stages of prostate cancer, 2) to use fusion proteins and siRNA to experimentally adjust expression levels of connexin isoforms, searching for effects on cell phenotype and behavior, and 3) to further identify connexin isoform binding-partners, using immunoprecipitation. Not only will these studies clarify connexin protein and gap junctional roles in prostate epithelial-to-mesenchymal transformation (EMT), but they may also reveal diagnostic, connexin "fingerprints" for each cancer stage, and suggest new candidate targets for therapy aimed at decreasing prostate cancer cell invasion and metastasis. Finally, these studies may provide a long-sought link between andropause and prostate cell behavior-between long-distance, hormonal regulatory mechanisms, and those far less-studied mechanisms that depend upon direct cell-cell contact. [unreadable] [unreadable]